International Journal of Biological Macromolecules

25 (1999) 21 29

Minireview
Microbial inclusions with special reference to PHA inclusions and
intracellular boundary envelopes

R. Clinton Fuller *
Department of Biochemistry and Molecular Biology, Uni6ersity of Massachusetts Amherst, MA 01003, USA

Received 9 September 1998; accepted 22 October 1998

Abstract

Polyhydroxyalkanoates (PHAs), in the form of metabolic storage reserves, are assembled in intracellular cytoplasmic inclusions,
often called granules. This review discusses both the structure and function of this assembly. In addition an overview of other
microbial cellular inclusions is presented. This is not a compilation of all such structures but a description of those that are similar
in many ways to either the structure or function of the PHA inclusions and are made up of monolayer envelopes and their storage
compounds. Not unique, such inclusions provide many similar examples which, in turn, provide useful analogies to the PHA
inclusions. A study of the PHA inclusions has been carried out in a comparative electron microscope examination and by protein
analysis of a number of organisms and E. coli transformants. 1999 Elsevier Science B.V. All rights reserved.

1. Introduction In 1925 Lemoigne [1,2] first described the presence of

Polyhydroxyalkanoates (PHAs), found in a wide va-
riety of free living bacteria, are stored as intracellular
inclusions, serving as carbon and energy sources for
various metabolic syntheses and growth. These
polyesters are biosynthetic, biodegradable and biocom-
patible thermoplastic elastomers of great potential for
industrial and medical use. Over the last decade, many
meetings and symposia have been held on PHAs; the
number of laboratories and investigators has at least
quadrupled since 1990. The first meeting of The Inter-
national Symposium of Biological Polyesters took place
in Sitges, Spain, in 1990. Gottingen followed in 1992;
Montreal in 1994; Davos, Switzerland in 1996; and now
Tokyo in 1998. During this time, two Gordon Confer-
ences and several meetings of The Bio/Environmental
Degradable Plastics Society have been held in the gen-
eral area of biosynthetic and biodegradable polyesters.
* Corresponding author. Tel.: +1-413-545-0328; fax: + 1-413-545-
3291.
E-mail address: rcfuller@biochem.umass.edu (R.C. Fuller)
poly(hydroxybutyrate PHB) in bacteria. But research in
this area was continued by only a few investigators
until new interest was awakened, in the 1960s and
1970s, by Merrick, Marchessault, Lundgren and their
co-workers [311]. These investigators made the initial
studies on the metabolism, enzymology, intracellular
inclusions and material properties of the polymer. One
of the more important discoveries at that time was
made by Wallen, White and associates [12,13] who
demonstrated that a number of the polyesters were
composed of units with varying chain lengths in a
variety of bacteria isolated from the environment. So
the polyesters were not simply repeating units of bu-
tyrate, but random copolymers of units with differing
chain lengths. The laboratories of Schlegel, Dawes,
Sinskey, Dennis and Witholt with their associates laid
the groundwork for future research on the cellular
enzymology and molecular genetics of these polyester
systems [1425]. These studies primarily involved the
use of Ralstonia eutropha (formerly Alcaligenes eutro-
phus) and Pseudomonas oleo6orans. Then, ICI in Eng-

0141-8130/99/$ - see front matter 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 1 4 1 - 8 1 3 0 ( 9 9 ) 0 0 0 1 1 - 2
22 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129
land developed scaled-up growth and production of the
PHB/PHV co-polymer, later selling the technology to
Monsanto. Without these early pioneers in the field of
PHAs, we would not be where we are in 1998.
As of 1998, over 100 PHAs have been described in
the literature [26]. The repeating unit composition of
the PHAs depends on the bacterial species and the
chain length of the carbon source fed during synthesis.
The chain length can range from three to 15 carbons. In
addition, many of the monomer precursors used for cell
growth and polymer synthesis can contain a wide vari-
ety of functionalized groups [27,28]. These modifica-
tions yield a chemically functional polymer that allows
further substitution and modification of the material
properties.
Since 1990, a number of comprehensive reviews have
given an excellent exposure to both the current state of
the science and the literature on microbial polyesters
through 1995 [26,29 35]. This review will not try in any
way to cover PHAs from microbes to materials but will
concentrate on the recent published literature that deals
with the in vivo or native PHA inclusion. Particular
attention will be paid to the nature and function of the
hydrophobic inclusion bodys boundary surface with
the hydrophilic bacterial cytoplasm.
2. Background
The eukaryotic cell is usually differentiated from the
prokaryote cell by its more highly evolved, compart-
mentalized nature. In eukaryotes the organelles are
complex, unit membrane-bound structures with varying
functions. True cellular membranes are defined as lipo
protein bilayers separating two water-containing com-
partments. Mitochondria and chloroplasts are utilized
for energy production. Genetic transcription, transla-
tion and ribosome synthesis occur in the nucleolus and
the nucleus. Liposomes, not bound by a unit membrane
and at a pH lower than the cytoplasm are used in the
metabolic degradation of stored polymers. The chloro-
plasts in higher plants and algae, as well as the mito-
chondria in all eukaryotic cells, all have an
ultrastructure consisting of an energy-transducing unit
membrane characterized by a lipoprotein bilayer. The
metabolic functions of the chloroplast and mitochon-
dria, which contain their own DNA and code for some
of their own metabolic activities, also are controlled
partially by the DNA of the nucleus. An example of the
highly developed intracellular coordination in eukary-
otic cells exists in the enzyme for the primary carboxy-
lation reaction in photosynthesis, ribulose bisphosphate
carboxylase (RUDP carboxylase). This protein consists
of two polypeptide subunits, one of which is coded for
in the chloroplast and the other in the nucleus. Assem-
bled within the chloroplast, this enzyme only functions
there. Clearly, highly complex signalling and cytoplas-
mic transport takes place between such eukaryotic
organelles.
Although all of the above metabolic and genetic
reactions occur in prokaryotes, the partition and com-
partmentalization of a unit membrane organelle, as a
rule, is not present. This does not mean that the
microbial world consists of cells full of soluble unorga-
nized metabolic systems. First of all, many bacterial
cells excrete enzymes that carry out metabolic activ-
itylargely polymeric degradationat a distance.
Also, the periplasmic space between the cell wall and
the unit cytoplasmic membrane carries out compart-
mentalized degradation equivalent to that of the eu-
karyotic lysosomes. In both types of cell, not only the
enzyme activity but also its intracellular organization
are important for intracellular turnover and efficient
metabolic regulation.
Prokaryotic cellular inclusions consist of a wide vari-
ety of structures: highly crystallized components as well
as amorphous or fluid ones not partitioned from the
cytoplasm by the lipoprotein bilayer unit membrane.
There are three general categories of prokaryotic inclu-
sions: structures of metabolic machinery; adjusters of
the environment; and producers of metabolic products
(reserves). These three classes of structures have been
described and reviewed by Shively in 1974 [36], by
Shively and co-authors in 1988 [37], and again by
Shively in 1998 [38]. This review will summarize only
briefly these classes in order to draw some comparisons
with the structure and function of several prokaryotic
inclusions, to the PHA inclusion body, primarily in
Pseudomonas oleo6orans, P. putida and recombinant E.
coli.
2.1. Prokaryotic inclusions as metabolic machinery
2.1.1. Carboxysomes
These structures, found in a variety of autotrophic
bacteria, photosynthetic bacteria and cyanobacteria,
are made up of crystalline polyhedral arrays or granules
that measure 40900 nm in diameter and consist of
eight proteins. The RUDP carboxylase, the enzyme for
autotrophic carbon dioxide fixation, makes up 50% of
the total complex. The structures are partitioned from
the cytoplasm by a 34-nm thick shell which consists of
several proteins, including four glycoproteins interfac-
ing with the cytoplasm. As with the PHA inclusion, no
unit membrane structure bilayer exists at the boundary.
The enzyme is internalized and attached to the outer
protein shell by the small subunit of the RUDP car-
boxylase enzyme. This structure is of unusual interest
since it accounts for up to 50% of the total protein on
earth! That the enzyme is organized and stored as a
crystalline protein assembly in prokaryotes allows rapid
mobilization and regulation for carbon dioxide fixation
and carbon metabolism [3638].
 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129 23

Fig. 1. Topological diagram of the chlorosomemembrane complex of Chloroflexus aurantiacus. This model is derived from the freeze-fracture
micrographs of Staehelin et al. [43], physicochemical parameters observed by Betti et al. [65], biochemical analysis by Feick and Fuller [41], and
Bchl-c primary and secondary structure by Wechsler et al. [66]. All of these diverse analyses complement and confirm each other. The chlorosome
contains the Bchl-c polypeptide arranged as a dimer in a tubular array of globular subunits (5.8 5.2 nm) with the seven interacting porphyrin
rings linearly arranged along the helix of the monomer. The baseplate B790 energy transfer polypeptide bridges the area between the chlorosome
and the cytoplasmic membrane. The transmembrane P865 reaction center and a hexameric Bchl-a antenna polypeptide complete the primary
light-harvesting and energy-transferring photosynthetic complex. From Ref. [37], copyright, Academic Press, 1988, and permission of authors.

2.1.2. Chlorosomes consisting of lipid and a protein envelope. After har-

Cohen-Bazire et al. [39] first described chlorosomes in
the green photosynthetic bacteria in the obligate anaer-
obic Chlorobium limocola. In 1966 the facultative green
bacteria Chloroflexus aurantiacus was described by Holt
et al. [40]. These chlorosomes are found only in the
family of the green photosynthetic bacteria. The ultra-
structure was described by Feick and Fuller [41] (Fig.
1). Green bacteria, including their structural aspects,
have been reviewed recently by Olson [42].
Located in the periphery of the cytoplasm, chloro-
somes are attached to but not derived from the cyto-
plasmic membrane. The chlorosome, as expressed in
Chloroflexus, consists of an organized array of rod-like
elements, 2552 nm in size. Three major proteins,
phospholipids, the antenna pigment bacteriochlorophyll
c (Bchl-c), and a baseplate protein attached to the
cytoplasmic membrane, make up this structure. Two of
the proteins exist on the surface, forming a lipoprotein
envelope boundary with the cytoplasm; the third takes
the form of linear, rod-like elements associated with the
antenna Bchl-c. Stachelin et al. have described the
chlorosome as expressed in the green sulfur bacterium
Chlorobium [43]. Thus the chlorosome is a light-harvest-
ing, non-unit membrane assembly in the cytoplasm,
vesting light energy in the form of excited state Bchl-c,
it passes this energized state to both the reaction center
Bchl-a and the electron transport system of the unit
cytoplasmic membrane. In that membrane, the energy,
coupled with an ATP synthase, is converted to ATP.
This cytoplasmic inclusion and its phospholipid
protein boundary envelope again is reminiscent of the
PHA inclusion structure.
2.2. Phycobilisomes
Though present in a few specialized eukaryotic
marine red algae and dinoflagellates, phycobilisomes
are expressed almost exclusively in the prokaryotic
Cyanobacteria. These structures consist of proteins co-
valently linked to open chain tetrapyroles (phycobillin
pigments) [37]. Like the chlorosome, they function as a
light-harvesting antenna protein complex that captures
light quanta and passes its excited state to a chlorophyll
containing unit membrane where ATP is produced. Fig.
2, taken from Giddings et al. [44], shows a diagram of
the phycobilisome assembly. It consists of a monolayer
lipo-protein envelope which acts as a boundary with the
cytoplasm.
24 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129

Fig. 2. Model for the cyanobacterial thylakoid membrane. The hemidiscoidal PBS, which usually occur as regularly spaced rows, are attached to
the stomal (protoplasmic) surface of the thylakoid membrane. Each PBS is in contact with two Photosystem II (P680) reaction centers. Also
shown, in regions not covered by the PBS, are the other three major photosynthetic complexes: the Photosystem I (P700) reaction center complex;
the plastoquinol plastocyanin oxidoreductase (cytochrome f,b6 complex, and the ATP synthase (CF0 CF1, coupling factor) complex. EF,
exoplasmic fracture face; PF, protoplasmic fracture face. Taken from Giddings et al. [44]. From Ref. [37], copyright Academic Press, 1988, and
permission of authors.

It is interesting to note that these highly proteina- 2.3.1. Polyphosphates

ceous structures, when degraded, also can serve as a
source of nitrogen and carbon for the cell during condi-
tions of nutrient deficiency [45]. Thus the phycobilisome
not only serves as a photosynthetic light-harvesting
mechanism for these cells, but also as a source of
growth substrates when light and nitrogen are deficient
in the environment. The prokaryotic system, again like
the PHA inclusion, is highly efficient for survival in a
variable environment (see Section 2.3.3).
2.3. Prokaryotic inclusions as metabolic reser6es
These inorganic inclusions are made up of polyphos-
phate granules which are linear polymers of orthophos-
phate linked by high energy phosphate bonds, identical
as an energy source to that in ATP. Although the status
of their boundary with the cytoplasm is not clear, they
act as a reserve phosphate for the organism when
starvation for that element occurs. They also can act as
a source of metabolic energy during degradation of the
polymer [37]. Polyphosphate crystalline granules often
occur in cells that accumulate PHAs.

This category of inclusions, occurring in a wide range
of bacteria, is both organic and inorganic. These inclu-
sions are expressed either as organized crystallized
granules naked in the cytoplasm or as amorphous
structures separated from the cytoplasm by a protein
monolayer. This monolayer often is associated with a
lipid or a carbohydrate. For these inclusions to be
bound by a true bilayer membrane is rare. Several
examples are described below.
2.3.2. Fluid sulfur globules
Although many organisms metabolize sulfur, only a
few accumulate and store this element as a metabolic
reserve. These organisms include members of the green
and purple photosynthetic sulfur bacteria, members of
the filamentous gliding sulfur bacteria and some species
of the colorless sulfur bacteria. The physical state of the
sulfur and its various inclusions have been reviewed
recently [37,4648].
 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129 25

Of interest for this review, four known structural

expressions of stored sulfur, the so-called fluid glob-
ules, exist in both the cytoplasm and periplasm of the
bacterial cell. This microbial storage inclusion is the
only one known to be expressed in several different
forms and in various parts of the cell. Fig. 3 from
Shively et al. describes these four types of sulfur accu-
mulations in the cell [37].
Many other prokaryote inclusions resemble the PHA
ones, in either structure of type or function. However,
the above examples point out that the operation of such
systems in microbial cells is common and has evolved
as an efficient survival mechanism for organisms in
environments of metabolic stress. The most common
characteristic of these systems is that, for the most part,
a protein (sometimes associated with a phospholipid or
carbohydrate) monolayer envelope partitions them
from the cytoplasm in the cell. This differs from the
characteristic lipoprotein bilayer membrane of all eu-
karyote cells which use true bilayer membrane systems
for energy production, active metabolic transport and
compartmentalization.
In addition to the above inclusion studies, some of
the following prokaryote inclusions have been reviewed
or published recently:
(a) Glycogen (polyglucose) as a storage product in
the cytoplasm [49].
(b) Magnetosomes. Iron storage and magnetotaxis.
Fig. 3. Model showing the four different types of internal sulfur
Protein envelope in cytoplasm. There are proteins asso-
deposits (S) found in bacteria. CW, cell wall; CM, cell membrane.
ciated with these inclusions when isolated, but the The abbreviations for each sulfur inclusion type are: CMEP, sulfur
ultrastructure of the iron complex is not known [50]. deposit located outside of cytoplasm in a pocket formed by invagi-
(c) Gas vesicles. Gas-filled buoyancy conferring cyto- nated cytoplasmic membrane (no other envelope observed); CMEP-
plasmic inclusions with a protein envelope consisting of ENV, sulfur deposit bound by a distinct (usually single-layered)
a single protein (Gyp protein) [51]. envelope located outside of cytoplasm in a pocket formed by invagi-
nated cytoplasmic membrane; UMC, sulfur deposit located in the
cytoplasm and enclosed in a unit-type membrane; SMC, sulfur de-
2.3.3. PHA inclusions posit located in cytoplasm and bound only by a single-layered
The first isolation and purification of PHA inclusion envelope. From Ref. [37], copyright Academic Press, and permission
bodies was carried out by Williamson and Wilkinson in of authors.
1958 [52] and refined in 1968 by the group associated
with Merrick [53]. The latter authors demonstrated that
the polymer was packaged with both protein and lipid
besides the PHB. The proteins demonstrated depoly-
merase enzyme activity and also implied polymerase
activity. Marchessault first proposed a model for the
system, and its basic premise of a micelle formation at
some point during assembly of the inclusion is still
appropriate [8].
Current studies on the chemical content structure
and enzymatic activities have been carried out primarily
with R. eutropha and P. oleo6orans. Our current under-
standing of the PHA inclusion system has been ad-
dressed in studies by Witholt with co-workers [54],
Steinbuchel et al. [55,56] and the laboratories of Fuller
and Lenz [5759]. The results, not always in agreement,
reflect the differences in organisms and growth condi- Fig. 4. Freeze-fracture micrograph of purified P. oleo6orans inclusions
tions. Steinbuchel and co-workers using R. eutropha as showing surface protein lattice.
26 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129

Fig. 5. Electron micrograph of polymer inclusions from several organisms. Native polymer inclusions purified from left to right: (a) P. oleo6orans;
(b) P. putida (BMO1); and (c) R. eutropha. A highly ordered structure exists on the inclusions for both pseudomonad species. This rigid
organization is not present in the inclusions from R. eutropha (uranyl acetate-negative stain). Magnifications of (a), (b) and (c) equal at 140 000.
From Ref. [57], copyright Elsevier, 1998, and permission of authors.

Fig. 6. A recombinant E. coli containing only the R. eutropha genes for polymer synthesis but no structural proteins are expressed (magnification,
136 800). From Ref. [57], copyright Elsevier, 1998, and permission of authors.

an experimental system, have proposed a model with a
predominantly lipid surface that includes polymerase
activity as well as protein-designated phasing. The func-
tion of the latter is not known. In the same paper, they
also described four classes of PHA inclusion proteins, a
description that certainly helps to define the types of
inclusion proteins for the various organisms. These
classes include: (1) PHA polymerases; (2) depoly-
merases; (3) phasins, which may play a structural role
at the polymercytoplasmic boundary; and (4) all other
proteins. How the last two groups interrelate is not
clear. Some of the latter proteins, though still referred
to as non-enzymatic surface proteins, may also play a
role defined for phasins as described below.
Research that relates to the PHA inclusion structure
in P. oleo6orans, P. putida and recombinant E. coli has
been published by the author of this mini-review and
co-workers [5759].
Fig. 4 is a freeze-fracture electron micrograph of P.
oleo6orans inclusion that shows the lattice-like array of
protein on the surface of the interphasing with the
cytoplasm.
Fig. 5 is a comparative view of the inclusions of P.
oleo6orans, P. putida and R. eutropha. Both pseu-
domonads show, not unexpectedly, a remarkable simi-
larity in their lattice-like surfaces. This similarity was
confirmed by Western blot analysis [35]. However, the
surface boundary of R. eutropha is less organized and
 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129
Fig. 7. PHA inclusions isolated from an E coli-double recombinant. Has genes for polymerase and synthetic enzymes and 24-kDa phasin protein
from Ralstonia (magnification, 136 800). From Ref. [57], copyright Elsevier, 1998, and permission of authors.
shows no lattice structure. Recently published research
[57,58] relates mostly to the PHA inclusion structures in
P. oleo6orans, P. putida and recombinant E. coli. How-
ever, other organisms show similar morphological char-
acteristics. Inclusions from both Nocardia corallina (not
shown) and Azetobacter 6inelandii (not shown) exhib-
ited boundary surface arrays which differed from the
Pseudomonads and Ralstonia. The surface arrangement
on Ralstonia in Fig. 5 agrees with those of Steinbuchel
et al. [56]. Although all the examined species contained
a characteristic protein boundary envelope, the struc-
tural arrangement and immunological characteristics
varied [57].
As described by Valentin et al. [58], an intrastructural
study of genetically altered E. coli has been carried out.
This work was based on the construction of transfor-
mants demonstrated by the work of Kidwell et al. [60]
and earlier genetic analyses of Huissman et al. [61].
Polymer inclusions were isolated from two different
transformants. The first is the E. coli HM174 which
harbors a gene plasmid pJ9238, including the poly-
merase gene (phaC) as well as the 3-ketothiolase and
acetoacetyl-CoA reductase genes from R. eutropha. The
second transformant harbors both plasmid pJM9238
and pBluescript KS%::phaP. The latter can produce the
polymerase as well as the small 24-kDa phasin protein
associated with the R. eutropha inclusion [61]. The
electron micrograph, shown in Fig. 6, is a transformant
expressing only the polymerase, biosynthetic ketothio-
lase and NADH reductase genes (PHACAB). These
cells exhibited small inclusions with a relatively unorga-
nized surface.
27
In contrast, as shown in Fig. 7, the double transfor-
mants expressing both the polymerase and the G24KD
phasin genes showed large PHA inclusions. Their en-
velopes were similar to the structure from R. eutropha
[55,57] but now expressed in E. coli.
Clearly, non-enzymatic boundary proteins (phasins)
of the inclusion envelope play an important role not
only in the organization of the inclusion, but also in
efficient production of in vivo PHA. The production of
PHA in genetically engineered cells of E. coli, higher
plants and yeast, using only the polymerase gene in the
transformants, has been consistently quite low. There-
fore, discovering that E. coli produces normal inclu-
sions only in the presence of both polymerase and the
phasin-expressing genes of R. eutrophus is encouraging.
It would be interesting to see if the same condition held
for other organisms not producing PHA, such as yeast
or higher plants. It may be that the envelope protein
genes are essential for optimal PHA production.
These same papers [57,58] reported mutant studies in
which wild-type P. putida, which accumulates medium
chain-length PHAs and, like inclusions in P. oleo6orans,
surrounds these inclusions with a highly organized
protein grid. The wild-type P. putida was subjected to
gene replacement mutagenesis which replaced various
regions of the PHA gene loci. Although one Mutant
c 42 retained the polymerase gene, phaC, and formed
intracellular PHA, it lacked the genes GA-1 and GA-2
for expressing the 18- and 43-kDa structural lattice
proteins. Structurally intact inclusions could not be
isolated from these cells. In contrast another experi-
28 R.C. Fuller / International Journal of Biological Macromolecules 25 (1999) 2129
ment demonstrated that structurally intact polymer in-
clusions could be isolated from a P. putida Mutant 201
which expressed the polymerase and the GA-2 gene
from P. putida which codes for the expression of the
outer inclusion-associated lattice protein. The mutant
produced 50% more PHA per cell mass than the mu-
tant lacking the inclusion boundary proteins [58].
Thus, information obtained from these ultrastruc-
tural and molecular genetic studies supports the follow-
ing conclusions. First, P. putida PHA synthesis, in the
absence of co-expressed genes for inclusion-associated
surface proteins, does not give rise to organized inclu-
sions; the inclusions are small, and the production of
PHAs is low. Second, as shown by the ultrastructural
study of PHA inclusions in E coli, double transfor-
mants (containing both the polymerase and a phasin
protein), the presence of that low-molecular weight
protein results not only in a remarkably more ordered
and defined inclusion boundary array, but also in larger
inclusions and greater production of PHAs. Macro-
molecular model assemblies of both R. eutropha
[55,62,63] and P. oleo6orans [64] PHA inclusions have
been proposed. Although the protein surfaces vary in
ultrastructure, they both appear valid for each organ-
ism and serve an important role in efficient PHA
production.
Acknowledgements
The author wishes to thank the US National Science
Foundation, The US Office of Naval Research and the
Johnson and Johnson Focused Giving Program for
financial support during the course of this research. My
colleagues Professor L.J.R. Foster, S.D. Goodwin and
R.W. Lenz at the University of Massachusetts were
invaluable during discussions and for their support as
well as encouragement throughout the research. Help
from Professor Jessup Shively of Clemson University
and John Stolz of Duquesne University in sorting out
the mysteries of microbial inclusions was essential! The
editorial assistance of Ms Veronica Morgan, during the
writing and assembling of the manuscript, was
invaluable.
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